Method for predicting the sensitivity to chemotherapy

ABSTRACT

Herewith described is a novel method for predicting the sensitivity towards chemotherapy, of a patient in need thereof, which comprises obtaining a blood sample from the patient and detecting the levels of blood glutathione (GSH) as a surrogate marker for glutathione-S-transferase (GST) activity in tumor tissues.

[0001] The present invention relates to the field of cancer treatmentand, more particularly, it relates to a method for predicting thesensitivity towards chemotherapy of a patient, by measuring glutathione(GSH) or GSH-related enzyme glutathione-S-transferase (GST) blood levelsof the said patient undergoing chemotherapeutic treatment.

[0002] The levels of glutathione (GSH) or (GST) are known in the art tobe correlated with the response to cytotoxic antitumor treatments sincehigh levels of GSH or GST confer resistance to several antitumor drugssuch as, for instance, alkylating agents (e.g. melphalan, chlorambucil,cyclophosphamide, ifosfamide mustards, BCNU), platinum complexes (e.g.cisplatin, carboplatin and oxaliplatin) and anthracyclines (e.g.doxorubicin, epirubicin, idarubicin and daunorubicin) [Biochem.Pharmacol 35: 3405-3409 (1986)].

[0003] Both GSH and GST are ubiquitously present in several humantissues such as, for instance, blood cells, plasma, serum circulatingblasts and pathologic (tumor) tissues.

[0004] See, for general references to GSH and GST, Cancer Res. 54:4313-4320 (1994); Brit. J. Cancer 72(2): 324-326 (1995); Drug DiscoveryToday 3:113-121 (1998).

[0005] GST, and most prominently GST-π, are present at high levels in apreponderance of tumor types. Increased levels of GSH and activity ofGST in comparison to normal tissues has been found in several tumortypes comprising, for instance, gastrointestinal tumors, uterine andovarian cancers, head and neck cancer, lung carcinomas, sarcomas, livertumors and haematological tumors [Cancer Res. 49:5225-5229 (1989);Clinical Reviews in Biochemistry and Molecular Biology 27(4.5):337-386(1992)].

[0006] GSH plays a crucial protective role against cellular injuryproduced by a number of toxic insults. Preclinical and clinical studieshave established a correlation between GSH/GST over expression andcancer or cancer response to chemotherapy.

[0007] Alterations of the GSH-based detoxification system (consisting ofGSH and GSH related enzymes, GSTs) have been also associated withvarying responsiveness to several antineoplastic agents.

[0008] So far, because of the low rate of responsiveness to conventionalchemotherapy in those tumors over expressing GSH/GST, the identificationof new markers predicting sensitivity to therapy is of utmostimportance.

[0009] Of additional importance was the requirement to identify thesenew predictive markers from a relatively non-invasive source, forinstance blood or blood component, to allow these predictive markers tobe readily analyzed for the evaluation of chemotherapy sensitivity.

[0010] We have now found that GST activity in tumor tissues is stronglycorrelated with blood GSH levels, hence indicating blood GSH levels as apossible surrogate marker for GST activity in tumor tissues.

[0011]FIG. 1: correlation between GST activity in tumor tissue and GSHlevels in matched whole blood specimens from lung cancer patients.

[0012]FIG. 2: correlation between GST activity in tumor tissue and GSHlevels in matched whole blood specimens from head and neck cancerpatients.

[0013] Therefore, it is a first object of the present invention a methodfor predicting the sensitivity towards chemotherapy of a patient in needthereof, which comprises obtaining a blood sample from the patient anddetecting the presence of blood glutathione (GSH) as a surrogate markerfor glutathione-S-transferase (GST) activity in tumor tissues.

[0014] According to the method of the invention, it is thus possible toidentify whether a given tumor is associated with GSH/GST overexpression, hence allowing the selection of the most suitable antitumortherapy.

[0015] It is therefore a further object of the invention a method forselecting the proper chemotherapeutic treatment for a patient in needthereof, which first comprises predicting his sensitivity towardschemotherapy by obtaining a blood sample from the patient, detecting thepresence of blood glutathione (GSH) as a surrogate marker forglutathione-S-transferase (GST) activity in tumor tissues, determiningwhether the blood GSH levels fall within a range indicative of apotential for the patient to exhibit de novo or later progression toresistance to chemotherapeutic agents, and selecting a suitable andeffective chemotherapeutic treatment.

[0016] In other words, once the blood levels of GSH being thus detectedare so high to indicate, for the patient, the possibility of exhibitingresistance to conventional chemotherapeutic agents, for instancealkylating agents, anthracyclines or platinum complexes, a suitable andeffective chemotherapeutic treatment, based on the above GSH levels,might comprise the administration of an antitumor agent which iseffective in the treatment of those tumors over expressing GSH/GST.

[0017] In this respect, the compoundN-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide(internal code PNU 166196), and pharmaceutically acceptable saltsthereof, recently appeared to be effective in the treatment of a tumorknown to be poorly responsive or resistant to conventional antitumortherapies and described in the literature as potentially over-expressingGSH/GST.

[0018] For a general reference to the above compound of formula

[0019] and to its effectiveness against tumors over expressing GSH/GSTsystem, see the international patent application WO 98/04524 and WO01/85144 (filed on Apr. 19, 2001 and claiming priority from UK patentapplication No. 0011059.3, filed on May 8, 2000), both in the name ofthe Applicant itself and herewith incorporated by reference.

[0020] Preferably, a suitable therapy could thus comprise theadministration to a patient in need thereof, of the proper amounts ofthe compound PNU 166196, for instance according to the administrationschedule reported in the international patent application WO 02/28389(claiming priority from U.S. Ser. No. 09/676,770, filed on Oct. 2, 2000)in the name of the Applicant itself and herewith incorporated byreference.

[0021] According to a preferred embodiment of the invention, the abovemethod for predicting the sensitivity towards chemotherapy could beadvantageously used in several tumor forms including, for instance,gastrointestinal tumors, uterine and ovarian cancers, head and neckcancer, lung carcinomas, sarcomas, liver tumors, pancreatic cancer,breast cancer, prostate cancer, melanoma and haematological tumors.

[0022] Even more preferably, the said tumor is selected from lung, headand neck cancer.

[0023] In addition, the above method may also be applied to select theproper antitumor therapy as a second line therapy, for instance once aprevious chemotherapy treatment, for example a first-line chemotherapytreatment with conventional antitumor agents, e.g. alkylating agents,platinum derivatives or anthracyclines, failed to give the expectedresults because of the occurrence, among other effects, of theaforementioned resistance effects.

[0024] Several methods are known in the art for the assay of GSH andrelated kits are commercially available.

[0025] According to the present invention, therefore, any commerciallyavailable kit for detecting GSH levels in blood samples may beconveniently employed.

[0026] In this respect, it is a further object of the invention the useof a kit for determining blood GSH levels as a surrogate marker for GSTactivity in tumor tissues.

[0027] With the aim of illustrating the present invention, withoutposing any limitation to it, the following experimental part is nowgiven.

Experimental Part

[0028] The following experimental part was used to demonstrate thestrong correlation existing between the GSH levels in blood versus theGST activity in tumor tissues, so as to render GSH detection in blood asa surrogate marker for GST levels in tumor tissues.

[0029] As formerly indicated, FIGS. 1 and 2 clearly show the abovecorrelations between GSH levels in blood of lung cancer patients andhead and neck cancer patients, with the GST activities in tumor tissuesof the said patients.

[0030] Tissue and blood samples from 29 patients with lung cancer(NSCLC) and 23 patients with head and neck cancer (SCC) were enrolled,as per the following table I. TABLE I Patient series Principalcharacteristics Head and neck cancer Lung cancer No. 23 29 Age 56(29-72) 67 (28-80) Sex 16 m - 7 f 24 m - 5 f Tumor type SCC 26 (NSCLC) 2 (lung adenocarcinoma)  1 (spino cell.)

Sampling Modalities

[0031] Tissue from primary or relapsed tumor. A sample (E 200 mg) oftumor tissue adjacent to the sample submitted for histologicalexamination was collected from each patient. Tissue samples were putimmediately in crushed ice. Samples were frozen in liquid nitrogenwithin 30 minutes (max 1 hour) from the excision.

[0032] Blood (before treatment of the primary tumor or at time offailure). Blood (15 ml) was collected in a pre-chilled syringe andprocessed as follows.

[0033] 3 ml were dispensed in K₃EDTA (or ACD-solution A) tubes andstored at −20° C. (whole blood).

Analytical Methods

[0034] GSH quantity GSH level in cytosol and whole blood samples wasmeasured by a commercially available GSH assay kit (Cayman, Ann Arbor,Mich., USA). This kit utilizes an enzymatic recycling method based onthe reaction between GSH and DTNB that produces a yellow colouredcompound (TNB). The rate of TNB production is directly proportional tothe concentration of GSH in the sample. Measurement of the absorbance ofTNB at 405 nm provides an accurate estimation of GSH in the sample.

[0035] Before assaying, samples were deproteinated with 10%metaphosphoric acid (MPA) to avoid interferences due to sulfhydrylgroups on the proteins in the assay. 50 μl of the deproteinated sample(whole or diluted 1:3 with kit Wash Buffer) were assayed in duplicateaccording to manufacturer's instructions. GSH concentration was measuredby comparison with a standard curve obtained by plotting the absorbanceat 25 min vs. GSH concentration (nmol/ml). Cytosol GSH levels werenormalised for protein content (nmol/mg).

[0036] GST activity. 10 μl of cytosol was analysed by a commerciallyavailable assay kit (Novagen, Darmstadt, Germany) according tomanufacture's instructions. The kit is designed in order to perform acolorimetric-enzymatic assay of glutathione S-transferase (GST). Thesample is combined with 1-chloro-2,4-dinitrobenzene (CDNB) substrate inthe supplied reaction buffer and the absorbance of the reaction ismonitored at λ=340 nm. The rate of change in A₃₄₀ is proportional to theamount of GST activity in the sample. The absorbance at 340 nm wasmonitored every 30 sec. over a period of 5 min for cytosol samples.

[0037] GST activity of all samples was compared with a standard (cytosolof human placenta) and was measured as U*/mg prot for cytosol sample.

[0038] *U=(dA/min of 10 μl placenta)/mg prot of placenta

Assay Validation

[0039] The validation of the methods was planned taking into account:sensitivity, specificity, precision (intra-assay, inter-assay,inter-batch), calibration range, reagent stability, and analytestability in different storage conditions.

[0040] GSH

[0041] The analytical sensitivity (evaluated as the mean+3 SD of 8replicates of the zero standard) was 0.33 nmol/ml.

[0042] Functional sensitivity was evaluated by plotting the imprecisionprofile of the method.

[0043] The minimum concentration with a coefficient of variation (C.V.)less then 10% was 0.4 nmol/ml.

[0044] Assay kit is based on a reaction between GST-reductase and DTNBthat reacts with all groups—SH contained in the sample. A highspecificity is expected since:—all thiol protein groups are removed bydeproteination;—GST-reductase is a specific enzyme for GSHsubstrate;—the reaction is monitored at λ=405 that is specific for GSH.No further confirmation experiments were thus performed.

[0045] Precision was evaluated by analysing, for 5 consecutive runs, aduplicate of whole blood. We obtained an inter-assay C.V. below 12%while the intra-assay C.V. was below 5% of variability (tables 5 and 6).

[0046] The calibration curve ranges between 0.6-40 nmol/ml.

[0047] All reagents must be stored at +4° C. until expiration dateindicated by manufacturers.

[0048] After opening, reagents are stable for 2 weeks at +4° C.

[0049] Samples and deproteinated samples are stable up to 6 months ifstored at −80° C. and −20° C., respectively.

[0050] GST Activity

[0051] Analytical sensitivity was evaluated by 8 replicates of the zerostandard and resulted 0.0055 U/ml.

[0052] Functional sensitivity was evaluated on 8 replicates of lowactivity sample. Since C.V. of replicates was less than 10% (9.2%) thecorresponding mean activity level (0.008 U of activity) was consideredas functional sensitivity.

[0053] Activity assay kit is based on an enzymatic reaction between GSTand CDNB, that is a specific substrate of the enzyme. Accordingly thespecificity of the method used is largely demonstrated in literature(Habig W. H., 1974; Smith D. B., 1988). We therefore did not performfurther confirmatory experiments.

[0054] Accuracy was evaluated with dilution test of a cytosol sample.Recovery was between 112% and 133%.

[0055] Precision was evaluated on 2 cytosol samples with two differentactivity levels. Four replicates of the samples were assayed on 5different runs. Inter and intra-assay C.V. were respectively under 9% ofvariability in high activity level sample and under 14% of variabilityin low activity level sample.

[0056] The calibration curve ranges between 0.01-0.4 dA/min

[0057] All reagents must be stored at −20° C. until expiration dateindicated by manufacturers. Samples are stable up to 6 months if storedat −80° C.

Results

[0058] GSH levels were measured in whole blood from 29 patients withlung cancer and 23 with head and neck cancer. Mean level in blood is 516nmol/ml (S.D.=117) in lung cancer and 428 nmol/ml (S.D.=97) in head andneck cancer. TABLE II GSH levels Summary Statistics Whole blood(nmol/ml) Overall mean 477 median 458 10°-90°% 350-620 n  52 pairedWilcoxon test <0.0001 (0.0001) Lung cancer mean 516 median 494 10°-90°%383-681 n  29 paired Wilcoxon test  0.0004 (0.0001) Head and neck cancermean 428 median 426 10°-90°% 317-566 n  23 paired Wilcoxon test   0.03(0.0532)

GST Activity

[0059] Total GST activity was measured in cytosol but not in plasmasample, because of low levels of the GST enzymes in this matrix. In factwe tested 21 plasma samples of 29 available lung cancer patients and 15plasma samples of 23 available head and neck cancer patients. GSTactivity was close to sensibility threshold of the method being notdetectable in 11/21 lung and 3/15 head and neck samples.

[0060] GST activity was measured in 29 tissue samples of lung cancer andin 22 of head and neck cancer. Mean activity is 1.72 U/Mg (S.D.=0.89) inlung cancer tissue. In head and neck, mean activity is 2.61 U/mg(S.D.=1.74). TABLE III GST activity Summary Statistics Cancer tissueU/mg Overall mean 2.1 median 1.72 10°-90°% 1.06-3.31 n 51 pairedWilcoxon test <0.0001 (0.0001) Lung cancer mean 1.72 median 1.3710°-90°% 0.87-2.97 n 29 paired Wilcoxon test  0.0002 (0.0001) Head andneck cancer mean 2.61 median 2.49 10°-90°% 1.11-3.42 n 22 pairedWilcoxon test   0.02 (0.0789)

CONCLUSIONS

[0061] The evaluated methods are reliable and robust for routine use intissue extracts (GST activity) and in whole blood (GSH level).

[0062] A highly significant positive correlation was found between wholeblood GSH and tissue GST activity.

[0063] In particular, the GST activity in cancer tissue vs. GSH level inwhole blood resulted to be correlated in lung cancer (r0.53, p=0.003,FIG. 1) and in head and neck cancer (r=0.89, p<0.0001; FIG. 2). TABLE IVGST activity in cancer tissue vs. whole blood GSH levels Tumor SpearmanCorrelation p value Lung 0.53 0.004 Head and neck 0.89 <0.0001

[0064] The above results clearly provide evidence that the GSH levels inblood samples of a cancer patient can be used as a surrogate marker forGST activities in tumor tissues, thus allowing to predict whether thepatient responsiveness to chemotherapy is associated with GSH/GST systemover expression.

1. A method for predicting the sensitivity towards chemotherapy, of apatient in need thereof, which comprises obtaining a blood sample fromthe patient and detecting the presence of blood glutathione (GSH) as asurrogate marker for glutathione-S-transferase (GST) activity in tumortissues.
 2. The method of claim 1 which allows predicting whether thegiven tumor is associated with GST over expression.
 3. The method ofclaim 2 wherein the tumor is selected from the group consisting ofgastrointestinal tumors, uterine and ovarian cancers, head and neckcancer, lung carcinomas, sarcomas, liver tumors, pancreatic cancer,breast cancer, prostate cancer, melanoma and haematological tumors. 4.The method of claim 3 wherein the tumor is selected from head, neck andlung cancer.
 5. A method for selecting the proper chemotherapeutictreatment for a patient in need thereof, which first comprisespredicting his sensitivity towards chemotherapy by obtaining a bloodsample from the patient, detecting the levels of blood glutathione (GSH)as a surrogate marker for glutathione-S-transferase (GST) activity intumor tissues, determining whether the blood GSH levels fall within arange indicative of a potential for the patient to exhibit de novo orlater progression to resistance to anticancer chemotherapeutic agents,and selecting a suitable and effective chemotherapeutic treatment basedon the above GSH levels.
 6. The method of claim 5 for selecting theproper chemotherapeutic treatment for a patient suffering of a tumorselected from the group consisting of gastrointestinal tumors, uterineand ovarian cancers, head and neck cancer, lung carcinomas, sarcomas,liver tumors, pancreatic cancer, breast cancer, prostate cancer,melanoma and haematological tumors
 7. The method of claim 6 wherein thetumor is selected from head, neck and lung cancer.
 8. The method ofclaim 5 which, based on the blood GSH levels, allows to select theproper chemotherapeutic treatment which may comprise the administrationto the patient in need thereof of the compoundN-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide(internal code PNU 166196) or a pharmaceutically acceptable salt thereofor, alternatively, of a conventional antitumor agent.
 9. The method ofclaim 8 wherein the conventional antitumor agent is selected from thegroup consisting of alkylating agents, anthracyclines and platinumderivatives.
 10. A method for treating a patient suffering from a tumorover-expressing the GSH/GST system, which first comprises predicting hissensitivity towards chemotherapy by obtaining a blood sample from thepatient, detecting the levels of blood GSH as a surrogate marker for GSTactivity in tumor tissues, determining whether the GSH levels fallwithin a range indicative of a potential for the patient to exhibit denovo or later progression to resistance to chemotherapeutic agents, andselecting a suitable and effective chemotherapeutic treatment based onthe above GSH/GST levels which treatment may comprise the administrationof an effective amount ofN-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide(internal code PNU 166196), or of a pharmaceutically acceptable saltthereof.
 11. The method of claim 10 wherein the tumor is selected fromthe group consisting of gastrointestinal tumors, uterine and ovariancancers, head and neck cancer, lung carcinomas, sarcomas, liver tumors,pancreatic cancer, breast cancer, prostate cancer, melanoma andhaematological tumors.
 12. The method of claim 11 wherein the tumor isselected from head, neck and lung cancer.
 13. Use of a kit fordetermining blood GSH levels as a surrogate marker for GST activity intumor tissues.